The present invention relates to diiminopiperazine derivatives and the use of these bioactive compounds in altering the metabolism and functions of sphingolipids, glycerolipids, and other biochemicals to affect diverse cell behaviors, including signal transduction, cell growth and survival.
Sphingolipids are a group of lipids found in all eukaryotic cells as well as some prokaryotes and viruses. Sphingosine and other long-chain (sphingoid) bases are the structural backbones of sphingolipids and have been found to affect diverse cellular systems when added to in vitro assays, cells, and in vivo when applied to skin, injected or fed in the diet. These affected systems include, but are not limited to, protein kinase C, Na+, K+-ATPase, phosphatidic acid phosphatase, phopholipases (including phospholipase D), retinoblastoma protein phosphorylation, and sphingosine-activated protein kinases. The inhibition of protein kinase C has been studied most thoroughly in vitro using mixed micellar assays of the purified enzyme as well as by evaluation of cellular functions dependent on this enzyme in platelets, neutrophils, HL-60 cells, and many other systems. Sphingosine inhibits protein kinase C by acting as a competitive inhibitor of activation by diacylglycerol, phorbol dibutyrate, and, for some isozymes, calcium, and also blocks activation by unsaturated fatty acids and other lipids. The exact mechanism by which sphingoid bases inhibit protein kinase C remains unknown. However, since protein kinase C binds to membranes through interactions with diacylglycerol and negatively charged phophatidylserine, sphingosine may be localized in regions of acidic lipids and block enzyme binding or activity. Modified forms of sphingosine (such as sphingosine 1-phosphate and N-acylsphingosines, or ceramides) are also biologically active.
Free sphingoid bases (sphinganine and sphingosine), sphingosine-1-phosphate, and ceramides are formed endogenously as lipid mediators. Sphingolipids have been studied as intracellular lipid messengers for agonists such as tumor necrosis factor, interleukin-1xcex2, platelet-derived growth factor, nerve growth factor, cytotoxic agents, and are involved in various forms of stress.
The changing of cells in culture to fresh medium has been found to induce a transient xe2x80x9cburstxe2x80x9d of sphingosine, sphinganine, and other bioactive lipids to levels that are sufficient to affect at least one signaling pathway, the membrane association and activity of protein kinase C. This increase in free sphinganine, as well as sphinganine-1-phosphate and dihydroceramide, upon the addition of new medium arises from de novo sphingolipid biosynthesis. In contrast, the increase in sphingosine (and sphingosine-1-phosphate and ceramide) arises mainly from the turnover of complex sphingolipids.
Because these sphingolipid metabolites are highly active and control important cell functions, which include, but are not limited to, inflammation, growth, cell differentiation and development, cell death, and aging, there is a need for methods and compositions for controlling or modulating sphingolipid metabolism. These methods and compositions will be useful in controlling the biological processes affected by sphingolipids and for the treatment of diseases that have disorders involving cell regulatory pathways. Furthermore, studies have also shown that other bioactive lipid metabolites change during the sphingosine xe2x80x9cburstxe2x80x9d (such as diacylglycerols), hence, agents that affect one cell signaling pathway may have important effects on other cell signaling systems, and have broad pharmacologic utility. In addition to the implications of these compounds for health, the ability to modulate cell behavior can have applications in industry for the production of biomolecules using cells in culture and other types of bioreactors.
Diimino-piperazine derivatives and their use as modulators of cell regulation are provided. These compounds are particularly useful for the inhibition of sphingolipid metabolism as evidenced by their ability to suppress the sphingosine xe2x80x9cburstxe2x80x9d that occurs when cells in culture are changed to new medium.
In a first preferred embodiment, the derivatives, including all stereo-, regio- and geometric isomers, have the following structural formula: 
wherein
R is independently H, alkyl, aryl, heteroaryl, xe2x80x94C(O)R, xe2x80x94C(NR)OR, xe2x80x94C(O)NR2, xe2x80x94C(NR)NR2, xe2x80x94C(NR)SR or xe2x80x94C(S)NR2;
R1, R2, R3 and R4 are independently H, R, (CH2)nOR, (CH2)nNR2, CH2O(CH2)nOR, or CH2O(CH2)nNR2 
where n=1 to 6; and
X and Y are independently O, NR1, S or CR12.
In a second preferred embodiment, the derivatives, including all stereo-, regio- and geometric isomers, have the following structural formula: 
wherein
R is independently H, alkyl, aryl, heteroaryl, xe2x80x94C(O)R, xe2x80x94C(NR)OR, xe2x80x94C(O)NR2, xe2x80x94C(NR)NR2, xe2x80x94C(NR)SR or xe2x80x94C(S)NR2;
R1, R2, R3 and R4 are independently H, R, (CH2)nOR, (CH2)nNR2, CH2O(CH2)nOR, or CH2O(CH2)nNR2 
where n=1 to 6;
X is independently O, NR1, S or CR12; and
Z is independently OR1, NR12, SR1.
In a third preferred embodiment, the derivatives, including all stereo-, regio- and geometric isomers, have the following structural formula: 
wherein
R is independently H, alkyl, aryl, heteroaryl, xe2x80x94C(O)R, xe2x80x94C(NR)OR, xe2x80x94C(O)NR2, xe2x80x94C(NR)NR2, xe2x80x94C(NR)SR or xe2x80x94C(S)NR2;
R1, R2, R3 and R4 are independently H, R, (CH2)nOR, (CH2)nNR2, CH2O(CH2)nOR, or CH2O(CH2)nNR2 
where n=1 to 6;
X is independently O, NR1, S or CR12; and
Z is independently O, NR1 or S.
In a fourth preferred embodiment, the derivatives, including all stereo-, regio- and geometric isomers, have the following structural formula: 
wherein
R is independently H, alkyl, aryl, heteroaryl, xe2x80x94C(O)R, xe2x80x94C(NR)OR, xe2x80x94C(O)NR2, xe2x80x94C(NR)NR2, xe2x80x94C(NR)SR or xe2x80x94C(S)NR2;
R1, R2, R3 and R4 are independently H, R, (CH2)nOR, (CH2)nNR2, CH2O(CH2)nOR, or CH2O(CH2)nNR2 
where n=1 to 6;
W is independently OR1, NR12, SR1, (CH2)nOR1, (CH2)nSR1 or (CH2)nNR12 
where n=1 to 6;
Z is independently OR1, NR12, SR1.
The most preferred diimino-piperazine derivative has the following chemical structure: 
The chemical name of the most preferred compound is 2,6-bis-(xcfx89-aminobutyl)-3,5-diimino-piperazine. This is a naturally occurring biochemical compound isolated from cells, particularly macrophages.
The derivatives can be synthesized using a series of reactions known to those skilled in the art, one example of which proceeds through a monomeric intermediate that is allowed to undergo a series of alkylation and subsequent deprotection reactions described in more detail below.
The diimino-piperazine derivatives alter sphingolipid metabolism as well as the metabolism of other bioactive mediators such as diacylglycerols, and are therefore useful when administered to patients with abnormalities that can be modified to achieve health benefit. Diseases that are in this category include, but are not limited to, inflammatory diseases, cancer, heart attack, strokes and other cardiovascular disease, AIDS, and neuronal and neuromuscular disease such as Alzheimer""s Disease and Parkinson""s Disease.
It is therefore an object of the present invention to provide a new category of bioactive compounds, namely diimino-piperazine derivatives, as naturally occurring or synthetic modulators of cell regulation.
It is a further object of the present invention to provide a treatment for diseases such as cancer that respond to modulation of cell regulation by administration of these compounds to patients.